Modified polypeptides with altered biological activity

ABSTRACT

The invention relates to novel modified polypeptides, with or without variations in noncoding regions, with altered biological activity. The invention discloses methods of preparing the modified polypeptides and methods of use.

GOVERNMENT SUPPORT

This invention was made with Government support under Contract No.N00014-90-J-1847 awarded by the Department of the Navy. The Governmenthas certain rights in the invention.

RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 08/756,134 filedNov. 26, 1996, issued as U.S. Pat. No. 5,747,446 on May 5, 1998, whichis a continuation-in-part of U.S. Ser. No. 08/216,259 filed Mar. 22,1994, issued as U.S. Pat. No. 5,580,853 on Dec. 3, 1996, the teachingsof which are incorporated herein by reference, in their entirety.

BACKGROUND OF THE INVENTION

Modification of naturally occurring polypeptides which have therapeuticvalue is often attempted in an effort to increase their biologicalactivity. Several methods have been employed to increase the biologicalactivity of therapeutic proteins. These methods often focus onincreasing the size of the therapeutic agents. For example, the size ofa protein can be increased through chemical conjugation with a reagentsuch as polyethylene glycol (PEG) (Knusli, C. et al., Brit. J. Haematol.82:654-663 (1992)). This procedure, also known as "PEGylation", has beenreported with several protein agents, first as a means to reduceantigenicity, but also as a way to increase biological activity.

Another method of increasing a protein's size is through chemicalcross-linking with another protein. For example, to increase theantigenicity of a protein, chemical cross-linking agents are used toconjugate the immunogenic protein to a carrier molecule such asimmunoglobulin or serum albumin.

However, the conjugation of chemical compounds or inert molecules to apolypeptide often results in a significant decrease of the overallbiological activity, and of selected biological activity of thepolypeptide, (Knusli, C., et al., Brit. J. Haematol., 82:654-663(1992)). These conjugations must be designed such that the resultingmodified polypeptide remains therapeutically efficacious and retains thedesired biological properties of the unmodified, wild type (i.e.,naturally-occurring) polypeptide (Satake, R., et al., Biochem. Biophys.Acta. 1038:125-129 (1990)).

Erythropoietin (EPO) is a glycoprotein hormone involved with the growthand development of mature red blood cells from erythrocyte precursorcells. It is a 166 amino acid polypeptide that exists naturally as amonomer. (Lin, F-K., et al. Proc. Natl. Acad. Sci. USA 82:7580-7584(1985)).

Several forms of anemia, including those associated with renal failure,HIV infection, blood loss and chronic disease can be treated with thishematopoietic growth factor. Erythropoietin is typically administered byintravenous or subcutaneous injection three times weekly at a dose ofapproximately 25-100 U/kg. Though quite effective, this form of therapyis very expensive. Estimates for the treatment of chronic dialysispatients have ranged from $8,000-10,000 per patient per year.

Another problem encountered in the practice of medicine when usinginjectable pharmaceuticals is the frequency at which those injectionsmust be made in order to maintain a therapeutic level of the compound inthe circulation. For example, erythropoietin has a relatively shortplasma half-life (Spivak, J. L., and Hogans, B. B., Blood, 73:90 (1989);McMahon, F. G., et al., Blood, 76:1718 (1990)), therefore, therapeuticplasma levels are rapidly lost, and repeated intravenous administrationsmust be made. An alternative route of administration is subcutaneousinjection. This route offers slower absorption from the site ofadministration, thus causing a sustained release effect. However,significantly lower plasma levels are achieved and, thus, a similarfrequency of injection, as is required with intravenous administration,must be used to get a comparable therapeutic effect. Therefore, it wouldbe advantageous to be able to modify therapeutically active proteins toincrease their biological activity and half-life which would result inless frequent injections or smaller doses of protein.

SUMMARY OF THE INVENTION

The present invention relates to modified polypeptides with increasedbiological activity, and methods of making these modified polypeptides.Increased biological activity is defined herein as a prolonged plasmahalf-life (i.e., a longer circulating half-life relative to thenaturally occurring polypeptide), or higher potency (i.e., requiring asmaller quantity relative to the naturally occurring polypeptide toachieve a specified level of biological activity). Increased biologicalactivity can also encompass a combination of the above-describedactivities, e.g., a modified polypeptide with higher potency that alsoexhibits a prolonged circulating half-life. In any case, because thepolypeptides have increased biological activity, the frequency withwhich they must be administered is reduced, or the amount administeredto achieve an effective dose is reduced. In any case, a reduced quantityof modified polypeptide would be necessary over the course of treatmentthan would be necessary if unmodified polypeptide were used.

Polypeptides encompassed by the present invention include anypolypeptides with therapeutic activity. Specifically encompassed by thepresent invention are cytokines, growth factors, and hormones whichinclude, for example, the following: Interferon-α, Interferon-β,Interferon-γ, Interleukin-1, Interleukin-2, Interleukin-3,Interleukin-4, Interleukin-5, Interleukin-6, Interleukin7,Interleukin-8, Interleukin-9, Interleukin-10, Interleukin-11,Interleukin-12, Interleukin-13, Interleukin-14, Interleukin-15,Interleukin-16, Erythropoietin, Colony-Stimulating Factor-1, GranulocyteColony-Stimulating Factor, Granulocyte-Macrophage Colony-StimulatingFactor, Leukemia Inhibitory Factor, Tumor Necrosis Factor, Lymphotoxin,Platelet-Derived Growth Factor, Fibroblast Growth Factors, VascularEndothelial Cell Growth Factor, Epidermal Growth Factor, TransformingGrowth Factor-β, Transforming Growth Factor-α, Thrombopoietin, Stem CellFactor, Oncostatin M, Amphiregulin, Mullerian-Inhibiting Substance,B-Cell Growth Factor, Macrophage Migration Inhibiting Factor,Endostatin, and Angiostatin. Descriptions of these proteins and assaysto assess biological activity can be found in "Human Cytokines: Handbookfor Basic and Clinical Research", Aggarwal, B. B., and Gutterman, J. U.,Eds., Blackwell Scientific Publications, Boston, Mass., (1992), which isherein incorporated by reference in its entirety.

More specifically, the present invention relates to modifiederythropoietin with increased biological activity, as defined above. Themodified erythropoietin of the present invention comprises wild typeerythropoietin that has been modified with a heterobifunctionalcross-linking reagent. A heterobifunctional cross-linking reagent isdefined herein as a reagent with two reactive groups that are capable ofreacting with and forming links, or bridges, between the side chains ofcertain amino acids, between amino acids and carboxylic acid groups, orvia carbohydrate moieties. In particular, the heterobifunctionalcross-linking reagents used in the present invention contain either acleavable disulfide bond group or a maleimido group.

The present invention also relates to multimeric polypeptidescomprising, for example, two, or more, erythropoietin moleculesconvalently linked together by one, or more, thioether bond(s). Theseerythropoietin multimers also exhibit increased biological activity. Thepresent invention further relates to methods of producing the modifiederythropoietin polypeptides with increased biological activity describedherein, and to methods of their use.

The modification of wild type erythropoietin with a heterobifunctionalcross-linking reagent containing a cleavable disulfide bond groupresulted in a modified erythropoietin with increased potency relative tounmodified wild type erythropoietin. Importantly, the disulfide bondgroup can be reduced to a free sulfhydryl group. The availability of afree sulfhydryl group on the erythropoietin polypeptide permittedfurther modification of erythropoietin to produce multimericerythropoietin with a prolonged circulating half-life relative to wildtype erythropoietin. The production of multimeric erythropoietin wasaccomplished by a method of chemically cross-linking two, or more,modified erythropoietin polypeptides. Briefly, the method is as follows.

A first erythropoietin derivative was produced by reacting wild typeerythropoietin with a heterobifunctional cross-linking reagentcontaining a cleavable disulfide bond group. The disulfide bond wasreduced to produce erythropoietin containing a free sulfhydryl group. Asecond erythropoietin derivative was produced by reacting wild typeerythropoietin with a heterobifunctional cross-linking reagentcontaining a maleimido group. The first and second erythropoietinderivatives were reacted together, thereby forming at least onethioether bond between the sulfhydryl and maleimido groups, thus forminga homodimer or homotrimer of erythropoietin. Surprisingly, thesemultimeric erythropoietin molecules exhibit biological activitycomparable to wild type erythropoietin. More importantly, theerythropoietin dimers showed a significantly prolonged circulatinghalf-life in vivo, relative to wild type erythropoietin.

Thus, as a result of the work presented herein, erythropoietin has nowbeen modified to produce erythropoietin compositions which exhibitincreased biological potency relative to wild type erythropoietin.Moreover, the modified erythropoietin of the present invention can bedimerized and trimerized with other modified erythropoietin molecules toproduce multimeric erythropoietin molecules with prolonged in vivocirculating half-lives. Although erythropoietin is used as the specificexample, it is understood that the instant invention described hereincan be used to produce multimers of any suitable polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the chemical structure of SPDP.

FIG. 1B shows the chemical structure of LC-SPDP.

FIG. 1C shows the chemical structure of sulfo-LC SPDP.

FIG. 2 shows the chemical structure of SMCC.

FIG. 3 is a histogram depicting the biological activity of the fractionscontaining homotrimers, homodimers and monomers of erythropoietincollected after high pressure liquid chromatography (HPLC).

FIG. 4 is a graphic representation of the results of a bioassaydemonstrating the increased in vivo half-life of the erythropoietindimer and monomer.

FIG. 5 is a graphic representation of the in vivo efficacy oferythropoietin dimers and monomers as measured by changes in hematocritsobtained before (Pre) and after (Post) the administration of 300 IU/kgprotein. The vertical bar represents the range of the highest and lowestvalue.

FIGS. 6A-D is a graphic representation of the in vivo efficacy oferythropoietin dimers (---) and monomers (▪---▪) as measured bychanges in hematocrits obtained before (Pre) and after (Post) theadministration of 300 (A and B) or 30 (C and D) IU/kg protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to modified polypeptides with increasedbiological activity, and methods of making and using these modifiedpolypeptides. Polypeptides suitable for modification by the methodsdescribed herein are polypeptides, preferably monomeric polypeptides,which do not contain any free sulfhydryl groups. Polypeptides of specialinterest are those polypeptides which interact with a cellular receptorto initiate cellular signaling events, for example, insulin anderythropoietin. Polypeptides encompassed by the present invention aretypically used as injectable therapeutic agents. If polypeptides withincreased biological activity are used as injectable therapeutic agents,the frequency of administration of these polypeptides can be reduced.

In one embodiment, the polypeptides described herein comprise wild type(e.g., naturally-occurring) proteins with therapeutic activity. Asdefined herein, therapeutic activity means the ability of a polypeptide,upon administration to a mammal, to alleviate, to any degree, oreliminate the deficiency or condition for which the mammal is beingtreated. Specifically encompassed by the present invention arecytokines, growth factors, and hormones which include, for example, theparticular proteins listed in the following paragraphs followed by theappropriate reference(s). Each of the references in the followingparagraphs is incorporated by reference in its entirety.

INTERFERON-α: Henco, K., et al., J. Mol. Biol., 185: 227-260 (1985).Pestka, S., et al., Ann. Rev. Biochem., 56: 727-777 (1987). Methods inEnzymology, Pestka, S., (Ed.), Academic Press, New York, N.Y., 119:3-14(1986).

INTERFERON-β: "Human Cytokines: Handbook for Basic and ClinicalResearch", Aggarwal, B. B. , and Gutterman, J. U. (Eds.), BlackwellScientific Publications, Boston, Mass. (1992).

INTERFERON-γ: Gray, P. W., et al., Nature, 298:859-863 (1982).Rinderknecht, E., et al., J. Biol. Chem., 259:6790-6797 (1984).

INTERLEUKIN-1: IL-1α: Furutani, Y., et al., Nucleic Acids Res.,143:167-3179 (1986). IL-1β: Clark, B. D., et al., Nucleic Acids Res.14:7897-7914 (1986).

INTERLEUKIN-2: Fujita et al., 1983., Williams, R. W., J. Biol. Chem.,260:3937-3940 (1985). Durand, D. B., et al., Mol. Cell Biol.,8:1715-1724 (1988).

INTERLEUKIN-3: Yang, Y. C., et al., Cell, 47:3-10 (1986). Manavalan, P.,et al., J. Protein Chem., 11:321-331 (1992).

INTERLEUKIN-4: Arai, N., et al., J. Immunol., 142:274-282 (1989).Redfield, C., et al., Biochem., 30:11029-11035 (1991). Powers, R., etal, Science, 256:1673-1677 (1992).

INTERLEUKIN-5: Azuma, C., et al., Nucleic Acids Res., 14:9149-9158(1986). Yokota, T., et al., Proc. Natl. Acad. Sci. USA, 84:7388-7392(1987). Parry, D. A. D., et al., J. Molec. Recognition, 1:107-110(1988).

INTERLEUKIN-6: Hirano, T., et al., Nature, 324:73-76 (1986). Van Snick,J., et al., Eur. J. Immunol., 18:193-197 (1988).

INTERLEUKIN-7: Goodwin, R. G., et al., Proc. Natl. Acad. Sci. USA,86(1):302-306 (1989).

INTERLEUKIN-8: Kusner, D. J., et al., Kidney International 39:1240-1248(1991).

INTERLEUKIN-9: Renauld, J-C., et al., J. Immunol., 144:4235-4241 (1990).Moeller, J., et al., J. Immunol. 144:4231-4234 (1990). Yang, Y. C., etal., Blood, 74:1880-1884 (1989).

INTERLEUKIN-10: Moore, K. W., et al., Science, 248:1230-1234 (1990).Fiorentino, D. F., et al., J. Exp. Med., 170:2081-2095 (1989).

INTERLEUKIN-11: Paul, S. R., et al., Proc. Natl. Acad. Sci. USA,87:7512-7516 (1990).

INTERLEUKIN-12: Wolf, S. F., et al., J. Immunol., 146:3074-3081 (1991);BLAST Database (www.ncib.nlm.nih.gov), accession number M65290.

INTERLEUKIN-13: Dolganov, G., Blood, 87:3316-3326 (1996).

INTERLEUKIN-14: Ambrus, J. L., et al., Proc. Natl. Acad. Sci. USA,90:6330-6334 (1993).

INTERLEUKIN-15: Meazza, R., et al., Oncogene, 12:2187-2192 (1996).Cosman, D., et al., Ciba Found. Symposium, 195:221-233 (1995).

INTERLEUKIN-16: Cruikshank, W. W., et al., Proc. Natl. Acad. Sci. USA,91:5109-5113 (1994).

ERYTHROPOIETIN: Jacobs, K., et al., Nature, 313:806-810 (1985); Lin,F.-K., U.S. Pat. No. 4,703,008 (1987); Powell, J. S., U.S. Pat. No.5,688,679 (1997).

COLONY-STIMULATING FACTOR-1: Kawasaki, E. S., et al., Science,230:291-296 (1985). Wong, G. G., et al., Science, 235:1504-1508 (1987).Ladner, M. B., et al., EMBO. J., 6:2693-2698 (1987). Cerretti, D. P., etal., Mol. Immunol., 25:761-770 (1988). "Colony Stimulating Factors",Dexter, T. M., et al. (Eds.), Marcel Dekker Publishers, New York, N.Y.pp. 155-176 (1990).

GRANULOCYTE-COLONY-STIMULATING FACTOR: Nagata, S., et al., Nature,319:415-418 (1986). Souza, L. M., et al., Science, 232:61-65 (1986).Parry, D. A. D., et al., J. Molec. Recognition, 1:107-110 (1988).

GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR: Miyataka, S., et al.,EMBO J., 4:2561-2568 (1985). Parry, D. A. D., et al., J. Molec.Recognition, 1:107-110 (1988). Manavalan, P., et al., 11:321-331 (1992).

LEUKEMIA INHIBITORY FACTOR: Moureau, J-F., et al., Nature, 336:690-692(1988).

TUMOR NECROSIS FACTOR: Nedwin, G. E., et al., Nucleic Acids Res.,13:6361-6373 (1985).

LYMPHOTOXIN: Nedwin, G. E., et al., J. Cell Biochem., 29:171-182 (1985).

PLATELET-DERIVED GROWTH FACTOR: Deuel, T. F., et al., J. Biol. Chem.,256:8896-8899 (1981). "Human Cytokines: Handbook for Basic and ClinicalResearch", Aggarwal, B. B. , and Gutterman, J. U. (Eds.), BlackwellScientific Publications, Boston, Mass. (1992).

FIBROBLAST GROWTH FACTORS: Abraham, J. A., et al., Science, 233:545-547(1986a).

VASCULAR ENDOTHELIAL CELL GROWTH FACTOR: Keck, P. J., et al., Science,246:1309-1312 (1989).

EPIDERMAL GROWTH FACTOR: Scott, J., et al., Science, 221:236-240 (1983).Gray, A., et al., Nature, 303:722-725 (1983).

TRANSFORMING GROWTH FACTOR-β: Derynck, R., et al., Nature, 316:701-705(1985). Scotto, L., et al., J. Biol. Chem., 265:2203-2208 (1990).

TRANSFORMING GROWTH FACTOR-α: Derynck, R., Cell, 54:593-595 (1988).

THROMBOPOIETIN: Sohma, Y., et al., FEBS Lett., 353: 57-61 (1994); BLASTDatabase (www.ncib.nlm.nih.gov), accession number D32046.

STEM CELL FACTOR: Williams, D. E., et al., Cell, 63:167-174 (1990).Copeland, N. G., et al., Cell, 63:174-183 (1990). Flanagan, J. G., etal., Cell, 63:185-194 (1990). Zsebo, K. M., et al., Cell, 63:213-224(1990). Martin, F. H., et al., Cell, 63:203-211 (1990). Zsebo, K. M., etal., Cell, 63:195-201 (1990). Huang, E., et. al., Cell, 63:225-233(1990). Anderson, D. M., et al., Cell, 63:235-243 (1990).

ONCOSTATIN M: Linsley, P. S., et al., Mol. Cell. Biol., 10:1882-1890(1990). Zarling, J. M., et al., Proc. Natl. Acad. Sci. USA, 83:9739-9743(1986). Malik, N., et al., Mol. Cell. Biol., 9:2847-2853 (1989).

AMPHIREGULIN: Plowman, G. D., et al., Mol. Cell. Biol., 10:1969-1981(1990). Shoyab, M., et al., Proc. Natl. Acad. Sci. USA, 85:6528-6532(1988).

MULLERIAN-INHIBITING SUBSTANCE: Cate, R. L., et al., Cell, 45:685-698(1986). Wallen, J. W., et al., Cancer Res., 49:2005-2011 (1989). Picard,J-Y., et al., Proc. Natl. Acad. Sci. USA, 83:5464-5468 (1986). Coughlin,J. P., et al., Mol. Cell. Endocrinol., 49:75-86 (1987).

B-CELL GROWTH FACTOR: Sharma, S., et al., Science, 235:1489-1492 (1987).

MACROPHAGE MIGRATION INHIBITORY FACTOR: Weiser, W. Y., et al., Pro.Natl. Acad. Sci. USA, 86:7522-7526 (1989).

ENDOSTATIN: O'Reilly, M. S., et al., Cell, 88:277-285 (1997).

ANGIOSTATIN: O'Reilly, M. S., et al., Cell, 79:315-328 (1994).

Many of the above described polypeptides can be grouped according tocommon structural motifs. For example, erythropoietin, interleukin-6,interleukin-4, interleukin-5, interferon-β,granulocyte-colony-stimulating factor and granulocyte-macrophagecolony-stimulating factor, have been shown to share a common structuralmotif characterized by a core of four alpha helices (for review see,Mott, H. R., et al., Cur. Opin. Struc. Biol., 5:114-121 (1995); Chaiken,I. M., et al., Trends in Biotech. 14:369-375 (1996)). Despite thediverse primary amino acid sequence of these polypeptides, the relatedtertiary structure predictions have lead to the identification of a newprotein superfamily which may share functional properties (e.g.,receptor binding) mediated, in part, by the alpha helices. Thebiological importance of four-helix bundle polypeptides in, for example,cell growth and differentiation, makes the design and production ofmultimeric forms of these polypeptides an important aspect of thepresent invention. Thus, specifically encompassed by the presentinvention are polypeptides characterized by four alpha helical bundles.

As described herein, polypeptides can be modified to increase theirbiological activity relative to the biological activity of the naturallyoccurring polypeptides. Increased biological activity, is defined hereinas a prolonged plasma half-life (i.e., a longer circulating half-lifethan the naturally occurring polypeptide), or higher potency (i.e.,requiring a smaller quantity than the naturally occurring polypeptide toachieve a specified level of biological activity). Increased biologicalactivity, as used herein, can also encompass a combination of the abovedescribed activities. For example, a modified polypeptide with higherpotency can also have an increased circulating half-life. In any case,because the polypeptides described herein have increased biologicalactivity, the frequency with which they must be administered can bereduced.

The polypeptides encompassed by the present invention are modified witha heterobifunctional cross-linking reagent. The heterobifunctionalcross-linking reagent can be attached to one, or more primary amine oramines, within the polypeptide. For example, the heterobifunctionalcross-linking reagent can be attached to the amino acid residue, lysineor to the alpha amino terminus of erythropoietin. Alternatively, forglycoproteins, the heterobifunctional cross-linking reagent can beattached to one, or more carbohydrate moiety, or moieties, in anoligosaccharide chain on the polypeptide.

The heterobifunctional cross-linking reagent is generally selected froma group of cross-linking reagents containing either a cleavabledisulfide bond group or a maleimido group. The addition of a disulfidebond group to a polypeptide also permits the design of a cross-linkingstrategy to produce multimeric polypeptides. The disulfide bond can becleaved by reaction with a known reducing agent, for example,dithiothreitol (DTT) which reduces the disulfide bond in thecross-linking reagent to produce a modified polypeptide derivativecontaining a free sulfhydryl (SH) group.

A second polypeptide derivative, capable of reacting with a freesulfhydryl group, is then produced by attaching a heterobifunctionalcross-linking reagent containing a maleimido group to the naturallyoccurring polypeptide. Again, the cross-linking reagent can be attachedto primary amines or carbohydrate moieties in the polypeptide. Theresulting polypeptide derivative containing a maleimido group is reactedwith the polypeptide derivative containing a reactive sulfhydryl groupresulting in a multimeric polypeptide molecule covalently linkedtogether by at least one thioether bond formed between the SH group andthe maleimido group.

Erythropoietin, a glycoprotein hormone involved with the growth anddevelopment of mature red blood cells from erythrocyte precursor cells,is a glycosylated polypeptide particularly suited for modification usingthe methods described herein. Erythropoietin is produced in the kidneyin response to hypoxia (e.g., red blood cell loss due to anemia) andregulates red blood cell growth and differentiation through interactionwith its cognate cellular receptor. Wild type erythropoietin is definedherein to include recombinant human erythropoietin (Powell, J. S., etal., Proc. Natl. Acad. Sci. USA, 83:6465-6469 (1986)), or naturallyoccurring erythropoietin which has been isolated and purified from blood(Miyake, T., et al., J. Biol. Chem., 252:5558-5564 (1977)) or sheepplasma (Goldwasser, E., et al., Proc. Natl. Acad. Sci. U.S.A, 68:697-698(1971)), or chemically synthesized erythropoietin which can be producedusing techniques well-known to those of skill in the art. For example,methods such as those described in Sytkowski and Grodberg (U.S. Pat. No.4,703,008); Sytkowski (U.S. Pat. No. 5,580,853); and Powell (U.S. Pat.No. 5,688,679), the teachings of which are incorporated herein byreference. Erythropoietin is a 166 amino acid polypeptide that existsnaturally as a monomer (Lin, F-K., et al., Proc. Natl. Acad. Sci. USA82:7580-7584 (1985)). The predicted secondary (McDonald, J. D., et al.,Mol. Cell. Biol., 6:842-848 (1986)) and tertiary (Biossel et al., J.Biol. Chem., 268:15983-15993 (1993)) structure of erythropoietin havebeen reported .

It was noted from the structure of wild type erythropoietin that thepolypeptide does not contain any free (reactive) sulfhydryl (SH) groups.(Boissel, J-P., et al., J. Biol. Chem 268:15983-15993 (1993)). Free SHgroups are useful for preparing conjugated proteins, such asradiolabeled antibodies (U.S. Pat. No. 4,659,839), or otherwisechemically modifying the polypeptide resulting in altered biologicalactivity of a polypeptide. A free sulfhydryl group can also play a rolein the binding of a polypeptide to its cellular receptor. For example,the polypeptide hormone, insulin, is covalently linked to its cellularreceptor via a disulfide exchange mechanism. (Clark, S. and Harrison, L.C., J. Biol. Chem., 258:11434-11437 (1983); Clark, S. and Harrison, L.C., J. Biol. Chem., 257:12239-12344 (1982)). Thus, a free sulfhydrylgroup can be critical to the biological activity of a polypeptide.Accordingly, a scheme was devised to modify wild type erythropoietin toattach a free sulfhydryl group.

In one embodiment of the present invention, wild type erythropoietin waschemically modified by the covalent attachment of a heterobifunctionalcross-linking reagent containing a cleavable disulfide bond group. Thecross-linking reagent was attached to a primary amine in theerythropoietin polypeptide. The attachment of a heterobifunctionalcross-linking reagent to wild type erythropoietin resulted inerythropoietin with increased potency relative to unmodifiederythropoietin.

Specifically, three different heterobifunctional cross-linking reagentswere used to produce modified erythropoietin with increased biologicalactivity. These cross-linking reagents were attached to one, or more,primary amine or amines in the wild type erythropoietin. Thecross-linking reagents were N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), "long chain" N-succinimidyl 3(2-pyridyldithio)propionate (LC-SPDP), wherein the length of the chain of SPDP isincreased with additional methyl groups, and sulfonated "long-chain"N-succinimidyl 3(2-pyridyldithio) propionate (sulfo-LC-SPDP) whereinLC-SPDP is sulfonated. SPDP (FIG. 1A), LC-SPDP (FIG. 1B) andsulfo-LC-SPDP (FIG. 1C) are commercially available cross-linking agents(Pierce Chemical Co., Rockford Ill.). SPDP, LC-SPDP and sulfo-LC-SPDPall contain an N-hydroxysuccimmidyl group to react with free aminogroups. In addition, these reagents all contain a disulfide bond groupthat can be further modified to form a reactive sulfhydryl group.

Another heterobifunctional cross-linking reagent that can be used tomodify wild type erythropoietin is a carbohydrate specific reagent thatattaches to carbohydrate moieties of glycosylated polypeptides. Thiscross-linking reagent, 3-(2-pyridyldithio) propionyl hydrazide (PDPH),contains an oxidized carbohydrate specific hydrazide and also contains acleavable disulfide bond group.

Wild type erythropoietin was modified with heterobifunctionalcross-linking reagents SPDP, LC-SPDP and sulfo-LC-SPDP as described indetail in Example 1. Briefly, erythropoietin was incubated in thepresence of specified concentrations of the chemical reagentN-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) so as to achievedifferent molar ratios of SPDP:EPO in solution. The unmodified wild typeerythropoietin and SPDP modified erythropoietin (SPDP-EPO) werebioassayed according to the method of Krystal, (Krystal, G., Exp.Hematol., 11:649-660 (1983)), which measures the effect oferythropoietin on erythropoiesis in intact mouse spleen cells. Theresults, shown in Table 1, demonstrate that SPDP-EPO exhibited anincreased biological activity relative to the control wild typeerythropoietin.

                  TABLE 1    ______________________________________    SPECIFIC ACTIVITY OF SPDP-MODIFIED ERYTHROPOIETIN    Reaction Mixture,                    Specific Activity    SPDP/EPO, mol/mol                    U/mcg    ______________________________________    0:1             200 ± 30    1:1             174 ± 20    3:1             340 ± 30    ______________________________________

Erythropoietin modified with sulfo-LC-SPDP (sulfo LC-SPDP-EPO), whichhas the advantage of increased solubility in aqueous solutions, was alsoprepared as described in Example 1. Incubation of erythropoietin in thepresence of sulfo-LC-SPDP at different molar ratios, followed bydialysis and biological assay revealed that sulfo-LC-SPDP modificationof erythropoietin resulted in a 530% increase in potency over theactivity of wild type erythropoietin, as shown in Table 2. Thus, thespecific activity of the erythropoietin was increased from 170 U/mcg forthe wild type erythropoietin to 900 U/mcg for the modifiederythropoietin prepared in the presence of 10 fold molar excess ofsulfo-LC-SPDP.

                  TABLE 2    ______________________________________    SPECIFIC ACTIVITY OF SULFO-LC-SPDP MODIFIED    ERYTHROPOIETIN    Reaction Mixture,    SULFO-LC-SPDP/EPO,                     Specific Activity    mols/mol         U/mcg    ______________________________________    Experiment #1     0:1             170 ± 20     5:1             220 ± 30    10:1             900 ± 70    30:1             600 ± 50    50:1             250 ± 30    100:1            350 ± 40    Experiment #2    0:1              200 ± 30    1:1              200 ± 40    2:1              370 ± 40    3:1              350 ± 40    6:1              380 ± 40    7:1              560 ± 50    10:1             900 ± 60    ______________________________________

LC-SPDP EPO was also prepared as described in Example 1. Although thebiological activity of this derivative was not evaluated, it isreasonable to believe that erythropoietin modified with LC-SPDP wouldalso exhibit increased biological activity due to its close structuralrelationship to SPDP and sulfo-LC-SPDP.

The chemically modified erythropoietin derivatives described above,which contained a cleavable disulfide bond group, permitted the designof a strategy to cross-link erythropoietin to form EPO-EPO dimers andEPO-EPO-EPO trimers with increased biological activity. These homodimers(EPO-EPO) and homotrimers (EPO-EPO-EPO) are "long-acting" erythropoietinproteins (also referred to herein as LA-EPOs). That is, these multimericerythropoietin derivatives exhibit a prolonged circulating half-liferelative to unmodified, erythropoietin.

The methods of preparing multimeric erythropoietin with increasedbiological activity are described in detail in Examples 2 and 3.Although erythropoietin is used as the specific example, it isunderstood that the methods described herein can be used to producemultimers (i.e., a polypeptide covalently cross-linked with one, ormore, identical polypeptides) of any suitable polypeptide.

Briefly, a first derivative of erythropoietin was prepared as describedin Example 1, by reacting erythropoietin with the compoundN-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to form SPDP-EPO.This reaction introduced an external disulfide bond group into theerythropoietin molecule. To form a free (or reactive) sulfhydryl group,SPDP-EPO can be exposed to a reducing agent, known to those of skill inthe art, to reduce the disulfide bond groups. As described in Example 2,SPDP-EPO was exposed to dithiothreitol (DTT), which reduces thedisulfide bond in the SPDP moiety to produce an erythropoietin moleculecontaining free SH groups, also referred to herein as SH-EPO.

A second erythropoietin derivative was produced by reactingerythropoietin with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, also known as SMCC (FIG. 2) to form SMCC-EPO.This reagent has an N-hydroxy succinimidyl (NHS) group at one end and amaleimido group at the other. The NHS group of SMCC reacts with freeamino groups in erythropoietin resulting in the formation of SMCC-EPO.The maleimido group of SMCC, now pointing outward from the SMCC-EPOderivative, reacts with free sulfhydryl groups found on SH-EPO.Therefore, when SH-EPO and SMCC-EPO are mixed together in solution, thereactive groups combine resulting in the formation of an EPO-EPO dimer,(i.e., one SH-EPO with one SMCC-EPO) or an EPO-EPO-EPO trimer (i.e., oneSMCC-EPO with two SH-EPOs, or two SMCC-EPOs with one SH-EPO) in whichthe modified erythropoietin polypeptides are covalently linked by atleast one thioether bond (e.g., one thioether bond in dimerized EPO andtwo thioether bonds in trimerized EPO). It is interesting to note thatSMCC-EPO, when tested in the Krystal bioassay, did not exhibit anyincreased biological activity relative to unmodified erythropoietin.

Alternatively, a heterobifunctional cross-linking reagent which containsa maleimido group to attach to carbohydrate moieties such as4-(4-N-maleimidophenyl) butyric acid hydrazide-HCl (MPBH) and4-(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide-HCl, can be used.

The first and second erythropoietin derivatives were reacted together asdescribed in detail in Example 2. The reaction resulted in the formationof multimeric erythropoietin, as well as unreacted monomericerythropoietin derivatives, which can be separated by high pressureliquid chromatography (HPLC), as described in Example 2. Theerythropoietin dimers comprised two erythropoietin polypeptides linkedby one or more thioether bonds. The erythropoietin trimers comprisedthree erythropoietin polypeptides, also linked by thioether bonds. Thetrimer can comprise two erythropoietin polypeptides, each containing afree sulfhydryl group which is linked with a third erythropoietinpolypeptide containing two or more, maleimido groups. Alternatively, theerythropoietin trimer can comprise one erythropoietin polypeptidecontaining two, or more, free sulfhydryl groups which is linked with twoerythropoietin polypeptides, each containing a maleimido group. Thepresence of EPO, EPO-EPO dimers and EPO-EPO-EPO trimers was confirmed byWestern blot analysis using antibodies specific for erythropoietin asdescribed in Sytkowski, A. J., and Fisher, J. W., J. Biol. Chem.,260:14727-14731 (1985).

Although the monomeric erythropoietin retained its biological activity,the erythropoietin dimers and trimers prepared under the conditionsdescribed in Example 2, with SH-EPO, did not exhibit biological activitywhen tested in the Krystal bioassay. Therefore, a second cross-linkingprotocol was designed in which a second type of SH-EPO derivative wasprepared using sulfo-LC-SPDP. This agent functions similarly to SPDP asoutlined above, however, it contains a spacer arm of several angstromsin length (e.g., wherein the number of CH₂ groups in the linear portionof the molecule is increased) resulting in increased physical separationof the species attached to its reactive ends. In particular,sulfo-LC-SPDP contains five methyl groups within the linear chain of themolecule, and is also sulfated to increase its aqueous solubility.

Multimeric erythropoietin produced using sulfo-LC-SPDP-EPO (SH-LC-EPO)as the first erythropoietin derivative was prepared, and separated byHPLC as described in detail in Example 2. HPLC fractions containing thetrimers, dimers and monomers were tested in the Krystal bioassay forbiological activity. Importantly, all three of these species, monomers,dimers, and trimers exhibited biological activity in the Krystal assay.(See FIG. 3).

Multimeric erythropoietin was also produced using heterobifunctionalcross-linking reagents containing a free sulfhydryl group attached tothe erythropoietin polypeptide and various heterobifunctionalcross-linking reagents containing a maleimido group, also referred toherein as "SMCC-like" reagents, as described in detail in Example 3. Asused herein, "SMCC-like" reagents are heterobifunctional cross-linkingreagents characterized by a N-hydroxy succinimidyl (NHS) group at oneend and a maleimido group at the other. As such they act in the samemanner as SMCC in that the NHS group of the "SMCC-like" reagents reactswith free amino groups in erythropoietin and the maleimido group of the"SMCC-like" reagents reacts with free sulfhydryl groups. SMCC-likereagents include, e.g., the following: GMBS, γ-maleimidobutyric acidN-hydroxysuccinimide ester; MMBS,m-maleimidobenzoyl-N-hydroxysuccinimide ester; EMCS, ε-maleimidocaproicacid N-hydroxysuccinimide ester; PMPBS, 4-(p-maleimidophenyl) butyricacid N-hydroxysuccinimide ester; and BMPS, β-maleimidoproprionic acidN-hydroxysuccinimide ester. Monomers, dimers and trimers produced withLC-SPDP and the SMCC-like reagents exhibited biological activity asmeasured in the Krystal assay.

The circulating half-life in vivo of erythropoietin homodimers wasdetermined as described in detail in Example 4. Monomeric and dimericerythropoietin was injected into rabbits, and blood samples wereanalyzed at 5 minutes and 2, 4, 6, 9, and 24 hours after injection. Asshown in FIG. 4, the biological activity of dimerized erythropoietin, asmeasured in the Krystal assay, was still evident 24 hours after theinitial injection, whereas the biological activity of monomericerythropoietin dropped off significantly earlier. Thus, the circulatinghalf-life of dimerized erythropoietin was more than three times longerthan wild type erythropoietin. The prolonged circulating half-life ofthe erythropoietin dimer may be due to its increased size relative tomonomeric erythropoietin, which would hinder its excretion from the bodythrough the kidney. Although the erythropoietin trimers were not assayedat this time, it is reasonable to predict that an EPO homotrimer wouldexhibit similar, or even longer circulatory half-life as the homodimersbecause a trimer has even greater size than a dimer. Theseerythropoietin dimers and trimers are also referred to herein aslong-acting erythropoietins (LA-EPOs).

The in vivo efficacy of erythropoietin dimers was determined asdescribed in detail in Example 5. Monomeric or dimeric erythropoietin(300 or 30 IU/kg) was injected into mice and hematocrits determined inblood samples obtained before (Pre) or after (Post) treatment.Erythropoietin was administered on days 1, 3 and 5; and hematocritsdetermined on day 8. As shown in FIG. 5 and FIG. 6A, injection of 300IU/kg of dimerized erythropoietin resulted in an increase in the meanhematocrit compared to animals injected with monomer. A ten foldreduction in the amount of erythropoietin injected (30 IU/kg) resultedin a similar pattern of efficacy as shown in FIG. 6C. Moreover, whenmice were treated with a single injection of monomer or dimer on day 1at a dose of either 300 IU/kg (FIG. 6B) or 30 IU/kg (FIG. 6D) thehematocrit of dimer treated mice remained elevated on day 8 unlike themonomer treated animals. Thus, the half-life and in vivo activity ofdimerized erythropoietin was augmented.

A single injection of 30 IU/kg of dimer increased hematocrits whereas asingle injection of 300 IU/kg monomer did not; therefore, erythropoietindimer is greater than ten fold as efficacious as monomer. Furthermore,on a molar basis the dimer dose was 38% that of the monomer due to thehigher specific activity of the dimer. Therefore, the in vivo activityof the dimer was greater than twenty six fold (10/0.38) higher than thatof the monomer.

The in vivo data described in detail in Examples 4 and 5 are significantin documenting biologically potent multimeric polypeptides with enhancedactivity and prolonged half-lives. Indeed, less frequent, for example,subcutaneous administration of polypeptides in a clinical setting can betherapeutically efficacious.

Preferred isomers of erythropoietin dimers and trimers can also beprepared. Nine primary amino groups have been identified in the humanerythropoietin molecule. At the amino terminus of erythropoietin is analpha amino group of alanine 1. Additionally, there are eight epsilonamino groups found on lysine 20, 45, 52, 97, 116, 140, 152 and 154. Whenusing LC-SPDP, SMCC, or SMCC-like reagents, one or more of these primaryamino groups is/are modified by the reagent.

Variations in the structure of the EPO/EPO dimer can alter theactivity/potency of the isoform. Altered biological activity as usedherein is defined as activity different from that of the wildtype orrecombinant polypeptide. For example, the activity of erythropoietin isto regulate the growth and differentiation of red blood cellprogenitors. Erythropoietin dimers or trimers, for example, can haveincreased activity relative to wildtype erythropoietin to regulategrowth and differentiation of red blood cell progenitor cells.Alternatively, the erythropoietin multimer proteins can have decreasedbiological activity relative to the wildtype erythropoietin.

Although the three-dimensional structure of erythropoietin is not known,teitiary structure predictions suggest that certain regions are held tobe important for receptor binding. Since the side chain of lysine,including its epsilon amino group, is hydrophilic, it is expected to beaccessible to solvent on the outside of the molecule and, therefore,could take part in erythropoietin receptor binding.

Chemical modification of such a lysine, for example, could decreaseactivity of the EPO/EPO dimer. Therefore, within the mixture of allpossible modifications, it is reasonable to expect that some moleculesare less active than others due to such unfavorable linkages. To put itanother way, some molecules are more active than others, that is, theyare preferred isomers. Another possibility is that steric factors couldposition the receptor binding domains of the dimer subunits in morefavorable steric or less favorable orientations. This could enhance orinhibit the likelihood that both binding domains of each dimer wouldbind simultaneously.

It is possible to modify amino groups preferentially so as to controlisomer structure. Several methods to control (target) modifications ofthe primary amino groups are described in Example 6.

As a result of the work described herein, modified polypeptides areprovided which exhibit increased biological activity. For example,erythropoietin modified with a heterobifunctional cross-linking reagentcontaining a cleavable disulfide bond group exhibited a 530% increase inbiological activity relative to wild type erythropoietin. This increasein biological activity indicates that an effective amount of modifiederythropoietin is substantially less than a comparable effective amountof wild type erythropoietin. The effective amount of modifiederythropoietin is defined herein as the amount of erythropoietinrequired to elicit an erythropoietic response, as indicated by increasedgrowth and/or differentiation of erythrocytic precursor cells. Forexample, if the typical effective dose of erythropoietin usedtherapeutically is 25 U/kg, then an effective dose of modifiederythropoietin can reasonably be as low as 5.0 U/kg to achieve the sameeffect.

Alternatively, the effective amount of multimeric polypeptide describedherein, with a prolonged circulating half-life, will require lessfrequent administration than an equivalent amount of wild typepolypeptide. For example, if an effective dose of erythropoietin istypically administered 3 times a week, multimeric erythropoietin withincreased biological activity will only need to be administered once aweek. In either case, a reduced quantity of erythropoietin modified witha heterobifunctional cross-linking reagent, or multimericerythropoietin, will be required over the course of treatment than isnecessary if wild type erythropoietin is used.

The modified polypeptides with increased biological activity describedherein can be used in place of wild type polypeptides whenever treatmentwith erythropoietin is called for. For example, modified erythropoietincan be used for treatment in an individual experiencing anemiaassociated with renal failure, chronic disease, HIV infection, bloodloss or cancer.

The polypeptides of the present invention are generally administered tohumans. Effective treatment with polypeptide requires maintaining atherapeutic blood level. This can be done by continuous administration,that is, by continuous intravenous injections, by discreet intravenousinjections, or by subcutaneous injection. The modified polypeptides ofthis invention can be employed in admixture with conventionalexcipients, i.e., pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral administration that do notdeleteriously react with the active derivatives.

Suitable pharmaceutically acceptable carriers include, but are notlimited to, water, salt solutions, alcohols, gum arabic, vegetable oils,benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such aslactose, amylose or starch, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid esters, hydroxymethycellulose,polyvinyl pyrrolidone, etc.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories.

It will be appreciated that the actual preferred amounts of activecompound in a specific case will vary according to the specific compoundbeing utilized, the particular compositions formulated, the mode ofapplication, the particular situs of application, and the organism beingtreated. Dosages for a given recipient will be determined on the basisof individual characteristics, such as body size, weight, age and thetype and severity of the condition being treated.

In addition, the modified polypeptides of the present invention, withincreased biological activity, can be used in any in vitro applicationin place of wild type polypeptide. For example, modified erythropoietincan be used in studies of erythropoietin receptor activity. It willagain be appreciated that the amount of modified erythropoietin withincreased biological activity needed to achieve desired results, (e.g.,increased hemoglobinization of red blood cell precursor cells) will besubstantially less than the amount of wild type erythropoietin requiredto achieve those desired results.

In another embodiment, the modified polypeptides described hereincomprise variant type polypeptides produced by modifications in 5'and/or 3' untranslated (UTR) or noncoding regions of the wildtype gene.Hereinafter, the term recombinant variant polypeptide will be used todescribe these molecules.

These recombinant variant polypetides can have altered biologicalactivity. Each individual polypeptide that comprises the homodimer orhomotrimers can itself have altered biological activity as compared tothe activity of the wildtype polypeptides. Altered biological activityis defined herein as activity different from that of the wildtype orrecombinant polypeptide. For example, the activity of erythropoietin isto regulate the growth and differentiation of red blood cellprogenitors. Recombinant erythropoietin variant polypeptides can haveincreased activity relative to wildtype erythropoietin to regulategrowth and differentiation of red blood cell progenitor cells.Alternatively, the erythropoietin variant polypeptides can havedecreased biological activity relative to the wildtype erythropoietin.

Mutations in noncoding regions of the gene (e.g., 5' untranslatedregions or UTR) can lead to differences in RNA secondary structure andtranslation is described, e.g., in Schultz, D. E., et al., J. Virol.70:1041-1049, 1996; Kozak, M., J. Mol. Biol. 235:95-110, 1994; andKozak, M., J. Biol. Chem. 266:19867-19870, 1991; Sytkowski, A. J., andGrodberg, J., U.S. patent application "Erythropoietin with AlteredBiological Activity", filed Feb. 3, 1998; and Stykowski, A. J., U.S.patent application "Production and Use of Recombinant Protein Multimerswith Altered Biological Activity," filed Feb. 3, 1998, the teachings ofwhich are incorporated herein by reference. As used herein, the termmutation refers to any alteration in the nucleic acid sequence encodinga polypeptide (e.g., a point mutation; the addition, deletion and/orsubstitution of one or more nucleotides).

Secondary structure has been shown to be a critical component indetermining the rates of translation efficiency of several proteins(Bettany, A. J., et al., J. Biol. Chem. 267:16531-16537, 1992; Kozak,M., J. Mol. Biol. 235:95-110, 1994). By implication, altered rates oftranslation can affect posttranslational modifications, for example,glycosylation patterns, and, thus, proper folding of the resultingprotein leading to changes in the chemistry, structure and function ofthe polypeptide. The recombinant variant polypeptides described hereinare unique in that they are composed of multimeric polypeptides producedby mutations in noncoding (5' and 3' UTR) regions of the gene.Mutations/deletions in the polypeptide noncoding regions can be made byusing any of a number of methods (e.g., site directed mutagenesis)familiar to those of skill in the art. For example, Sambrook, et al.,"Molecular Cloning: A Laboratory Manual", (1989); Ausubel, et al.,"Current Protocols in Molecular Biology", (1995); and Powell (U.S. Pat.No. 5,688,679), the teachings of which are incorporated herein byreference, amy be used.

The present invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLE 1 SPDP-EPO DERIVATIVE WITH HIGHER POTENCY

Three different heterobifunctional cross-linking reagents containingcleavable disulfide bond groups have been used to produce erythropoietinderivatives with increased biological activity. These agents areN-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), "long-chain"N-succinimidyl 3-(2-pyridyldithio) propionate, wherein the length of thechain of SPDP is increased with additional methyl groups (LC-SPDP), andsulfonated "long-chain" N-succinimidyl 3-(2-pyridyldithio) propionate(sulfo-LC-SPDP). Modified erythropoietin polypeptides were prepared asfollows.

Recombinant human erythropoietin was produced by expression of the humanerythropoietin gene in stably transfected BHK (baby hamster kidney)cells (Powell, J. S. et al., Proc. Nat. Sci. Acad. USA, 83:6465-6469(1986) and purified using standard laboratory techniques. The purifiedprotein was then incubated in the presence of specified concentrationsof the chemical reagent N-succinimidyl 3-(2-pyridyldithio) propionate(SPDP), dissolved in dimethyl sulfoxide, so as to achieve molar ratiosof 0:1, 1:1 and 3:1 (SPDP:EPO) in solution. After incubation overnightat room temperature, the solutions were dialyzed against phosphatebuffered saline to remove unreacted SPDP.

The wild type erythropoietin and modified erythropoietin (SPDP-EPO)samples were evaluated for biological activity according to the methodof Krystal. (Krystal, G., Exp. Hematol., 11:649-660 (1983)). Briefly,the bioassay of Krystal measures the effect of erythropoietin on intactmouse spleen cells. Mice are treated with phenylhydrazine to stimulateproduction of red blood cell precursor cells in the spleen. Aftertreatment, the spleens are removed, intact spleen cells are carefullyisolated and incubated with various amounts of wild type erythropoietinor the modified erythropoietin described herein. After an overnightincubation, ³ H thymidine is added and its incorporation into cellularDNA is measured. The amount of ³ H thymidine incorporation is indicativeof erythropoietin-stimulated DNA synthesis in erythroid precursor cellsvia interaction of erythropoietin with its cellular receptor. Theresults demonstrate that SPDP-EPO exhibited an increased biologicalactivity relative to the wild type erythropoietin, and that thisincrease in activity was proportional to the molar ratio of SPDP:EPO inthe reaction mixture.

Additionally, wild type erythropoietin was modified using sulfo-LC-SPDP,a compound which has the advantage of increased solubility in aqueoussolutions. Incubation of erythropoietin in the presence of sulfo-LC-SPDPat the previously described molar ratios followed by dialysis andbiological assay revealed that sulfo-LC-SPDP modification oferythropoietin resulted in an increase in potency of approximately 530%.The specific activity of the erythropoietin was increased from 170 U/mcgfor the nonderivatized material to 900 U/mcg for the materialderivatized in the presence of 10 fold molar excess of sulfo-LC-SPDP.

EXAMPLE 2 LONG-ACTING MULTIMERIC ERYTHROPOIETIN DERIVATIVES

To prepare the first SH-EPO derivative, 50 ug of human erythropoietinobtained as described in Example 1, was incubated in the presence offive-fold molar excess of N-succinimidyl 3-(2-pyridyldithio) propionate(SPDP) obtained from Pierce Chemical Company. After incubation at roomtemperature for sixteen hours, the solution was dialyzed againstphosphate buffered saline. The modified erythropoietin was then exposedto 1 mM DTT to reduce the disulfide bond in SPDP resulting in one, ormore, free sulfhydryl group(s) on the erythropoietin molecule.

The second erythropoietin derivative, SMCC-EPO, was prepared as follows.A second 50 ug portion of human erythropoietin was incubated in thepresence of five-fold molar excess of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). After a sixteen hour incubation atroom temperature, the solution was dialyzed against phosphate bufferedsaline.

The SH-EPO and SMCC-EPO were mixed together in phosphate buffered saline(20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) at roomtemperature for 90 minutes, and dialyzed against PBS. The mixture wasthen subjected to size exclusion HPLC chromatography on TSK 250, in PBS,room temperature, at 1 ml/min. The polypeptides were subjected to SDSpolyacrylamide gel electrophoresis, electrophoretic transfer tonitrocellulose, and Western blotting using anti-erythropoietinantibodies according to Sytkowski, A. J., and Fisher, J. W., J. Biol.Chem., 260:14727-14731 (1985). The results showed that the protocolsucceeded in the formation of two higher molecular weight species oferythropoietin corresponding to erythropoietin dimers and trimers.However, upon assay in the Krystal bioassay, the erythropoietin dimersand trimers produced with SPDP-SH-EPO did not exhibit any biologicalactivity.

Thus, the protocol was revised to use LC-SPDP-EPO as the firstderivative, 50 ug of recombinant human erythropoietin was incubated inthe presence of three-fold molar excess of LC-SPDP for sixteen hours atroom temperature. The material was then dialyzed and treated with 1 mMDTT resulting in SH-LC-EPO. SMCC-EPO was prepared as described above.

These two species were mixed together in solution and the mixture wassubjected to size exclusion HPLC on TSK3000SW. Three erythropoietinprotein species were detected with elution times of 10.2, 9.1, and 7.2minutes respectively. The 10.2 minute elution time was known fromprevious experiments to be that of wild type erythropoietin monomer.Therefore, the more rapid elution times of 9.1 and 7.2 minutescorresponded to dimers and trimers, respectively. The fractionscontaining the erythropoietin dimers and trimers were collected andassayed in the Krystal bioassay. Importantly, as shown in FIG. 3, upontesting in the Krystal bioassay, the erythropoietin homodimers andhomotrimers exhibited biological activity.

EXAMPLE 3 CROSSLINKING ERYTHROPOIETIN USING LC-SPDP AND SMCC-LIKEREAGENTS

Multimers of erythropoietin were also produced using LC-SPDP-EPOderivatives and EPO derivatives produced by reaction with SMCC-likereagents. The five SMCC-like cross-linking reagents were:

(1) GMBS, γ-maleimidobutyric acid N-hydroxysuccinimide ester;

(2) MMBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester;

(3) EMCS, ε-maleimidocaproic acid N-hydroxysuccinimide ester;

(4) PMPBS, 4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester;and

(5) BMPS, β-maleimidoproprionic acid N-hydroxysuccinimide ester.

All of these cross-linking reagents are commercially available, e.g.from Sigma Chemical Co., St. Louis, Mo. The chemical structures of thesecross-linkers are shown in Table 3.

                  TABLE 3    ______________________________________    CHEMICAL STRUCTURES OF "SMCC-LIKE"    CROSS-LINKING REAGENTS    ______________________________________    a. GMBS; γ-maleimidobutyric acid N-hydroxysuccinimide ester;    b. MMBS; m-maleimidobenzoyl-N-hydroxysuccinimide ester;    2    c. EMCS; ε-maleimidocaproic acid N-hydroxysuccinimide ester;    3    d. PMPBS; 4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester;    4    e. BMPS; β-maleimidoproprionic acid N-hydroxysuccinimide ester;    5    f. SMCC; succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate;    6    ______________________________________

To prepare LC-SPDP, 20 μg of human erythropoietin obtained as describedin Example 1, was incubated in the presence of ten-fold molar excess of"longchain"N-succinimidyl 3-(2-pyridyldithio) propionate (LC-SPDP)obtained from Pierce Chemical Company. The incubation occurred in sodiumphosphate 20 mM, sodium chloride 100 mM, pH 7.0 (PBS) at 23° C. for 30min. To stop the reaction, excess PBS at 4° C. was added to the mixture(final volume, 0.5 ml) and then dialyzed for at least 6 h at 4° C.against PBS (3×1.0 L). Finally, DTT (final concentration, 1 mM) wasadded to the mixture for 10 min to reduce the disulfide bond in LC-SPDP,resulting in one, or more, free sulfhydryl group(s) on theerythropoietin molecule.

The second erythropoietin derivative, SMCC-like EPO, was prepared asfollows. Aliquots of human erythropoietin (20 μg) were incubated with aten-fold molar excess of each of the five SMCC-like reagents listedabove and allowed to react. After a 30 min incubation at 30° C. in PBS,the reaction was stopped by adding excess PBS at 4° C. (final volume,0.5 ml). The mixture was then dialyzed for at least 6 h at 4° C. againstPBS (3×1.0 L).

Equal molar amounts of LC-SPDP EPO and each of the five SMCC-like EPOwere placed in five, separate dialysis bags and dialyzed against PBSovernight at 4° C. (1 L). The mixture from each of the dialysis bags wasthen individually subjected to size exclusion HPLC chromatography. Asize exclusion HPLC chromatography column, Progel TSK-3000 SW_(XL) (7.8mm I.D.×30 cm) and guard column, Progel TSK SW_(XL) (4.0 cm×6.0 mm I.D.)were equilibrated with 100 mM sodium phosphate, 150 mM sodium chloride,pH 7.0. 400 μl (16 μg of total EPO) of monomer/dimer/trimer mixture(e.g. EPO:LC-SPDP+EPO:GMBS) was separated on the equilibrated column ata flow rate of 1.0 ml/min and 0.2 ml fractions were collected. Theelution profile was monitored at 280 nm. Bovine serum albumin (finalconcentration, 2 mg/ml) was added to each fraction to stabilize thedimers/trimers and monomers. Elution profiles of the cross-linked EPOmultimers(e.g., EPO:LC-SPDP+EPO:GMBS; EPO:LC-SPDP+EPO:MMBS; EPO:LC-SPDP+EPO:EMCS; EPO:LC-SPDP+EPO:PMPBS; EPO:LC-SPDP+EPO:BMPS; andEPO:LC-SPDP+EPO:SMCC) were similar to those shown in FIG. 3 forEPO:LC-SPDP+EPO:SMCC multimers.

Ten μl of each HPLC fraction was diluted in 490 μl of bioassay medium(78% α-MEM, 20% FBS, 0.1 mM β-mercaptoethanol,1×penicillin/streptomycin/fungizone) and sterilly filtered through 0.2μm filters. Further final dilutions of 100×, 500× and 5000× were made ofthe fractions in bioassay medium and assayed for activity using theKrystal in vitro assay, as previously described. Fractions containingmonomeric EPO, dimerized EPO and trimerized EPO all exhibited biologicalactivity in the Krystal assay.

Western blot analysis was also performed on the HPLC fractions asfollows. Ten μl of each fraction was electrophoresed on a 10% SDSpolyacrylamide gel and transferred to nitrocellulose at 25 V for 18 h at4° C. in 25 mM Tris, 192 mM glycine and 10% methanol. The membranes wereblocked with 20 mM Tris-HCl, 500 mM NaCl, 0.1% Tween-20 (TBST)+10%Non-fat dry milk overnight with rocking at 4° C. They were then rinsed2× with TBST, washed 1× for 15 min, 2× for 5 min each, with TBST. Themonoclonal EPO antibody AE-7A5 (28 μl Ab in 50 ml TBST/5% dry milk) wasplaced over the membranes and rocked at 23° C. for 1 h. They were washedas above followed by incubation with goat anti-mouse IgG (Cappel,diluted 1000× in TBST/5% dry milk). Washing was carried out as abovewith additional 2× for 5 min each. Bands were detected using the ECLdetection reagents from Amersham. Equivolumes of solutions 1 and 2 weremixed and 10 ml of the mixture placed over each membrane. After 1 minthe membranes were wrapped in Saran Wrap brand plastic wrap and exposedto X-ray film. Fractions containing monomeric EPO, dimerized EPO andtrimerized EPO all specifically reacted with the anti-EPO antibodies.

EXAMPLE 4 IN VIVO TESTING OF MULTIMERIC ERYTHROPOIETINDERIVATIVES-HALF-LIFE DETERMINATIONS

A group of New Zealand white rabbits were injected intravenously eitherwith wild type monomeric erythropoietin or with dimerized LA-EPO, asprepared in Example 2, at 0.4 mg/ml in PBS. Blood samples were obtainedat 5 minutes and 2, 4, 6, 9, and 24 hours and measured the circulatingerythropoietin by the Krystal in vitro biologic assay. The results shownin FIG. 4 indicate that the in vivo half-life for monomeric wild typeerythropoietin was approximately seven hours, as expected frompreviously published reports. The in vivo half-life of LA-EPO however,was prolonged beyond the twenty-four hour period of the experiment asshown in FIG. 4.

EXAMPLE 5 IN VIVO EFFICACY OF MULTIMERIC ERYTHROPOIETINDERIVATIVES-EFFECTS ON HEMATOCRIT

Groups of three to four C57BL/6J mice (8-10 week old females) wereinjected subcutaneously either with wild type monomeric erythropoietinor with dimerized EPO, as prepared in Example 3, at 300 (FIG. 5; FIG. 6Aand 6B) or 30 (FIG. 6C and 6D) IU/kg body weight in 0.5 ml of PBS. Thebiological activity of the wild type and dimerized erythropoietin wasverified, prior to use, by in vitro bioassay according to theart-established procedures (Krystal, G., Exp. Hematol., 11:649-660(1983)).

Mice received either three injections (days 1, 3, and 5) or a singleinjection on day 1. The hematocrits of blood samples, obtained fromindividual mice before (Pre) and after (Post) treatment by retro-orbitalvenous plexus puncture, were determined. The rationale for treating onegroup of animals three times in the same week is based on the routineadministration of erythropoietin by subcutaneous or intravenousinjection to human patients three times weekly.

The mean hematocrit of dimer-treated mice injected with 300 IU/kg atdays 1, 3 and 5 increased from 46.9% (Pre-treatment) to 56.1% on day 8(Post-treatment) for a mean absolute increase of 9.2% (FIG. 5, Dimers).In contrast, the mean hematocrit of mice treated with monomer increasedfrom 46.9% (Pre-treatment) to 51% on day 8 (Post-treatment) for a meanabsolute increase of 4.1% (FIG. 5, Monomers). Therefore, treatment ofmice with dimerized erythropoietin at a concentration of 300 IU/kgresulted in a 2.2-fold increase in hematocrits compared to monomertreated animals. These data are duplicated in FIG. 6A.

A similar response pattern documenting the in vivo differences inmonomers and dimers was observed in mice injected with a ten fold lowerconcentration of erythropoietin (30 IU/kg). In animals receivinginjections on days 1, 3, and 5, the mean hematocrit of mice injectedwith monomer did not appreciably change (47.1% Pre-treatment to 48.2%Post-treatment for a 1.1% mean absolute increase; FIG. 6C ▪---▪)compared to animals injected with dimer (44.9% Pre-treatment to 50.0%Post-treatment for a mean absolute increase of 5.1%; FIG. 6C ---).

Of particular interest, was the observation that hematocrits remainedevaluated at day 8 in mice receiving a single dose of dimer on day 1(FIG. 6B and 6D). The mean hematocrit of mice injected with the highdose (300 IU/kg) of dimer increased from 46.6% to 50.3%, for a meanabsolute increase of 3.7% (FIG. 6B ---), unlike monomer treatedanimals where no appreciable change in hematocrit was observed (47.4%pre-treatment; 47.2% post-treatment; FIG. 6B ▪---▪). Similarly, the meanhematocrit of mice injected with the low dose (30 IU/kg) of dimerincreased from 47.2% to 49.9%, for a mean absolute increase of 2.7%(FIG. 6D ---), unlike monomer treated animals (48.6% pre-treatment;47.0% post-treatment; FIG. 6D ▪---▪). These data document that a singleinjection of erythropoietin dimer, unlike the monomeric form, canmaintain to an increase in the hematocrit of a mammal.

The in vivo efficacy as measured by a sustained increase in hematocritsis consistent with the prolonged plasma half-life of the dimeric form orerythropoietin (FIG. 4). Moreover, the data indicate that on a per unitbasis (molar or molecular mass) dimers can be administered lessfrequently with beneficial therapeutic results.

EXAMPLE 6 METHODS OF PREPARING AND PURIFYING PREFERRED ISOMERS OF EPODIMERS

ALTERING THE pH OF THE REACTION

The pKa's of alpha amino groups and of the epsilon amino group are 9.69and 10.53, respectively, but this is determined for free amino acids insolution. In contrast, when the amino acid is part of a polypeptide,these pKa's can vary greatly due to surrounding structures such as otheramino acid side chains. This means that within a given protein such aserythropoietin, each of the epsilon amino groups of the eight lysinescan have a different pKa. Lowering the pH of the reaction causesionization (protonation) of the NH₂ group to form a NH₃ ⁺ group, thusreducing its reactivity with the succinimidyl moiety of LC-SPDP or SMCC.

PROTECTING (BLOCKING) THE AMINO GROUP FROM THE MODIFYING REAGENT

A number of means can be used to protect amino acid side chains fromchemical modification. For example, site specific antibodies directedtoward certain regions of the amino acid sequence could be used. Bindingthe antibody to the erythropoietin prior to chemical modification wouldgreatly reduce or eliminate modification of those amino groups that formpart of the antigenic determinant or are sterically restricted by thebulky immunoglobulin molecule. A series of site specific antipeptideantibodies to erythropoietin covering numerous domains, some of whichinclude lysine residues have been made, as described in Sytkowski, A. J.and Donahue, K. A., J. Biol., 262:1161 (1987).

In addition to antibody protection, reversible chemical modification ofamino groups can be employed. Using this method, the protein is reactedwith a reversible modifying reagent such as maleic anhydride. Certainamino groups can be modified, thus preventing subsequent modificationwhen reacted with LC-SPDP, SMCC, or SMCC-like reagents. Following thesecond modification, the protecting group is removed with an additionalchemical reaction at low pH. This method can result in selectivemodification of unprotected amino groups.

A third means of protecting amino groups is specifically directed towardthe alpha amino terminal alanine 1. Instead of expressing the mature EPOprotein, the gene can be engineered so that additional amino acidsequence is expressed upstream of alanine 1. This can be engineered soas to include an enzymatic cleavage site immediately upstream ofalanine 1. Then, following modification with LC-SPDP or with SMCC, theupstream peptide sequence can be enzymatically cleaved, releasing themature EPO protein with an unmodified alpha amino group at alanine 1.

SIDE CHAIN TARGETING DUE TO PHYSICOCHEMICAL PROPERTIES AND/OR PHYSICALCHARACTERISTICS OF THE MODIFYING REAGENT

The physicochemical properties of the modifying reagent can cause it toselectively interact with certain amino groups of the protein. A classicexample of this type of effect is seen in the modification of horseliver alcohol dehydrogenase with iodoacetic acid. Reacting the enzymewith iodoacetic acid results in the highly specific modification ofcysteine 46, despite the fact that the enzyme contains numerous otherfree sulfhydryl groups. This specificity is due to the fact thatnegative charge interacts avidly with a positive charge on the arginylresidue adjacent to cysteine 46. This interaction directs theiodoacetate to this area of the enzyme resulting in a highly selectivemodification of cysteine 46.

With respect to the modifiers used to produce EPO dimers, the negativecharge on sulfo-LC-SPDP or sulfo-SMCC can reasonably similarly directthe modifying reagent to a positive charge. Additionally,nonpolar/hydrophobic moieties in the modifiers such as the cyclohexaneportion of SMCC can target the reagent to lysine residues adjacent tohydrophobic nonpolar amino acids.

SH-EPO and maleimido EPO monomers, modified preferentially on certainamino groups, can reasonably result in the production of site specificdimer isomers using the methods of producing dimers described herein. Alist of these isomers is presented in Table 4.

                  TABLE 4    ______________________________________    PRODUCTION OF SITE SPECIFIC DIMER ISOMERS                    Covalently Bonded to Maleimido    SH-EPO Modified at                    Modified EPO at    ______________________________________    Alanine 1       Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    Lys 20          Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    Lys 45          Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    Lys 52          Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    Lys 97          Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    Lys 116         Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    Lys 140         Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    Lys 152         Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    Lys 154         Alanine 1, lys 20, lys 45, lys                    52, lys 97, lys 116, lys 140, lys                    152, lys 154    ______________________________________

In addition to these possible dimer isomers, it is reasonable to expectthat favored trimer isomers also can be produced using these methods.

There are several methods that can be utilized to separate and purifythe EPO monomers that had been modified selectively as described above.These methods include reverse phase HPLC (RP-HPLC), ion exchangechromatography (e.g., DEAE or CM) and affinity chromatography onimmobilized EPO receptor. Each of these are described in detail below.

REVERSE PHASE HPLC (RP-HPLC)

The combination of linker polarity plus that of the surrounding aminoacid sidechains will determine the interaction of the modified EPOmonomer with the RP matrix and solvent. This will lead tochromatographically discrete behavior and specifically modified monomerscan be isolated.

ION EXCHANGE CHROMATOGRAPHY SUCH AS DEAE OR CM

Similarly, modification of specific amino groups will alter interactionof the charged EPO with both cation and anion exchangers.

AFFINITY CHROMATOGRAPHY ON IMMOBILIZED EPO RECEPTOR

EPO receptor protein can be expressed recombinantly, purified and linkedcovalently to a matrix such as agarose. This affinity matrix can then beused to isolate monomers with the highest affinity for the receptor, andsimultaneously to exclude monomers with low or absent receptor binding.

The methods described above for isolation of modified monomers can beapplied to dimer and trimer isomers as well. Additionally, sizeexclusion chromatography is available for isolation of modified dimersand trimers. The different conformation of the dimers and trimers willlead to molecules exhibiting different average stokes radii resulting indifferential behavior on high resolution size exclusion HPLC.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

What is claimed is:
 1. A biologically active polypeptide homodimercomprising two polypeptides covalently linked by at least one thioetherbond, wherein:a) the first polypeptide comprises a polypeptide with aheterobifunctional cross-linking reagent containing a free sulfhydrylgroup attached to the polypeptide; and b) the second polypeptidecomprises a polypeptide with a heterobifunctional cross-linking reagentcontaining a maleimido group attached to the polypeptide and at leastone thioether bond forms between the free sulfhydryl group of the firstpolypeptide and the maleimido group of the second polypeptide,where eachpolypeynide of the homodimer comprises a four alpha helical bundlepolypeptide.
 2. The polypeptide homodimer of claim 1, wherein thepolypeptide has altered biological activity.
 3. The polypeptidehomodimer of claim 1 wherein the heterobifunctional cross-linkingreagent is selected from the group consisting of: N-succinimidyl3-(2-pyridyldithio) propionate, "long chain" N-succinimidyl3-(2-pyridyldithio) propionate, sulfonated "long chain" N-succinimidyl3-(2-pyridyldithio) propionate, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, "long chain" succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfonated "long chain"succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate,γ-maleimidobutyric acid N-hydroxysuccinimide ester,m-maleimidobenzoyl-N-hydroxysuccinimide ester, ε-maleimidocaproic acidN-hydroxysuccinimide ester, 4-(p-maleimidophenyl)butyric acidN-hydroxysuccinimide ester, and β-maleimidoproprionic acidN-hydroxysuccinimide ester.
 4. A composition comprising a polypeptidehomodimer according to claim 1 and a pharmaceutically acceptablecarrier.
 5. The polypeptide homodimer of claim 1 wherein theheterobifunctional cross-linking reagents of a) and b) are attached toone, or more, primary amine or amines in the polypeptides.
 6. Thepolypeptide homodimer of claim 1 wherein the polypeptide is glycosylatedand the heterobifunctional cross-linking reagents are attached to one,or more, carbohydrate moiety or moieties in the glycosylatedpolypeptides.
 7. A method of preparing a biologically active polypeptidehomodimer according to claim 1, comprising the steps consisting of:a)preparing a first polypeptide derivative by reacting polypeptide with aheterobifunctional cross-linking reagent containing a cleavabledisulfide bond group, under conditions sufficient to introduce thecleavable disulfide bond group into the polypeptide, thereby producing afirst polypeptide derivative containing a cleavable disulfide bond; b)cleaving the disulfide bond group contained in the first polypeptidederivative, thereby producing a first polypeptide derivative containinga free sulfhydryl group; c) preparing a second polypeptide derivative byreacting polypeptide with a heterobifunctional cross-linking reagentcontaining a maleimido group, under conditions sufficient to introducethe maleimido group into the polypeptide, thereby producing a secondpolypeptide derivative containing a maleimido group; and d) reacting thefirst polypeptide derivative containing a free sulfhydryl group with thesecond polypeptide derivative containing a maleimide group, underconditions sufficient to form a thioether bond between the freesulfhydryl group and the maleimido group resulting in the cross-linkingof the first and second polypeptide derivatives, thereby producing amodified polypeptide comprising a first and second polypeptidederivative,wherein each polypeptide of the homodimer comprises a fouralpha helical bundle polypeptide.
 8. The polypeptide homodimer of claim7, wherein the polypeptide has altered biological activity.
 9. Thepolypeptide homodimer of claim 7 wherein the heterobifunctionalcross-linking reagent is selected from the group consisting of:N-succinimidyl 3-(2-pyridyldithio) propionate, "long chain"N-succinimidyl 3-(2-pyridyldithio) propionate, sulfonated "long chain"N-succinimidyl 3-(2-pyridyldithio) propionate, succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, "long chain"succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfonated"long chain" succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, γ-maleimidobutyric acid N-hydroxysuccinimideester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, ε-maleimidocaproicacid N-hydroxysuccinimide ester, 4-(p-maleimidophenyl)butyric acidN-hydroxysuccinimide ester, and β-maleimidoproprionic acidN-hydroxysuccinimide ester.
 10. The method of claim 7 wherein theheterobifunctional cross-linking reagents of step a) and step c) reactwith one, or more, primary amine or amines in the polypeptide.
 11. Themethod of claim 7 wherein the polypeptide is a glycosylated polypeptideand the heterobifunctional cross-linking reagents of step a) and step c)react with one, or more, carbohydrate moiety or moieties in theglycosylated polypeptide.
 12. A composition comprising a polypeptidehomodimer according to claim 7 and a pharmaceutically acceptablecarrier.
 13. A biologically active polypeptide homotrimer comprisingthree polypeptides covalently linked by thioether bonds, wherein;a) thefirst and second polypeptides comprise polypeptides with aheterobifunctional cross-linking reagent containing a free sulfhydrylgroup attached to each polypeptide; and b) the third polypeptidecomprises a polypeptide with a heterobifunctional cross-linking reagentcontaining two, or more, maleimido groups attached to the polypeptideand the thioether bonds form between the tree sulfhydryl group of thefirst and second polypeptides and the maleimido groups of the thirdpolypeptide,wherein each polypeptide of the homotrimer comprises a fouralpha helical bundle polypeptide.
 14. The polypeptide homotrimer ofclaim 13, wherein the polypeptide has altered biological activity. 15.The polypeptide homotrimer of claim 13 wherein the heterobifunctionalcross-linking reagent is selected from the group consisting of:N-succinimidyl 3-(2-pyridyldithio) propionate, "long chain"N-succinimidyl 3-(2-pyridyldithio) propionate, sulfonated "long chain"N-succinimidyl 3-(2-pyridyldithio) propionate, succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, "long chain"succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfonated"long chain" succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, γ-maleimidobutyric acid N-hydroxysuccinimideester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, ε-maleimidocaproicacid N-hydroxysuccinimide ester, 4-(p-maleimidophenyl)butyric acidN-hydroxysuccinimide ester, and β-maleimidoproprionic acidN-hydroxysuccinimide ester.
 16. A composition comprising a polypeptidehomodimer according to claim 13 and a pharmaceutically acceptablecarrier.
 17. A biologically active polypeptide homotrimer comprisingthree polypeptides covalently linked by thioether bonds, wherein:thefirst polypeptide comprises a polypeptide with a heterobifunctionalcross-linking reagent containing two, or more, free sulfhydryl groupsattached to the polypeptide; and b) the second and third polypeptidescomprise polypeptides with a heterobifunctional cross-linking reagentcontaining a maleimido group attached to each polypeptide and thethioether bonds form between the free sulfhydryl groups of the firstpolypeptide and the maleimido group of the second and thirdpolypeptides,wherein each polypeptide of the homodimer comprises a fouralpa helical bundle polypeptide.
 18. The polypeptide homotrimer of claim17, wherein the polypeptide has altered biological activity.
 19. Thepolypeptide homotrimer of claim 17 wherein the heterobifunctionalcross-linking reagent is selected from the group consisting of:N-succinimidyl 3-(2-pyridyldithio) propionate, "long chain"N-succinimidyl 3-(2-pyridyldithio) propionate, sulfonated "long chain"N-succinimidyl 3-(2-pyridyldithio) propionate, succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, "long chain"succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfonated"long chain" succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, γ-maleimidobutyric acid N-hydroxysuccinimideester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, ε-maleimidocaproicacid N-hydroxysuccinimide ester, 4-(p-maleimidophenyl)butyric acidN-hydroxysuccinimide ester, and β-maleimidoproprionic acidN-hydroxysuccinimide ester.
 20. A composition comprising a polypeptidehomodimer according to claim 17 and a pharmaceutically acceptablecarrier.